Efficient CRISPR-Cas9-mediated gene ablation in human keratinocytes to recapitulate genodermatoses: modeling of Netherton syndrome

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches

Análisis sectorial del sistema de franquicia español

El objetivo del presente trabajo es realizar un análisis, desde el punto de vista del franquiciador, en dos de los principales sectores de actividad de la franquicia como son los de Hostelería y Restauración, y el de Confección, Moda y Complementos con objeto de determinar las similitudes y diferencias en el arquetipo de franquiciador de cada sector. Este análisis se hará a partir de una base datos (año 2017) de 359 franquiciadores (221 franquicias del sector de hostelería y restauración; 138 franquicias del sector de confección, moda y complementos), analizándose los años de contrato, la inversión inicial, el canon de entrada, el canon de publicidad, los royalties, los establecimientos propios y franquiciados, la población mínima necesaria, el tamaño mínimo del local y la disponibilidad de portal web. Los resultados muestran que el sector condiciona el arquetipo de franquiciador, existiendo diferencias en los dos sectores analizados

Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations inCOL7A1 causing recessive dystrophic epidermolysis bullosaare amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeteddeletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA(sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved veryefficient targeted deletion of COL7A1 exon 80 in recessivedystrophic epidermolysis bullosa (RDEB) patient keratinocytescarrying a highly prevalent frameshift mutation. This ex vivonon-viral approach rendered a large proportion of correctedcells producing a functional collagen VII variant. The effectivetargeting of the epidermal stem cell population enabled longterm regeneration of a properly adhesive skin upon graftingonto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sitesdid not reveal any unintended nuclease activity. Our strategycould potentially be extended to a large number of COL7A1mutation-bearing exons within the long collagenous domainof this gene, opening the way to precision medicine for RDEB.The study was mainly supported by DEBRA International, funded by DEBRA Austria (grant termed as Larcher 1). Additional funds came from Spanish grants SAF2017-86810-R (to M.D.R.) and PI17/01747 (to F.L.) from the Ministry of Economy and Competitiveness and Instituto de Salud Carlos III, respectively, both co-funded with European Regional Development Funds (ERDF) ERA-NET E-RARE JTC 2017 (MutaEB) and CIBERER (grant termed as Murillas- TERAPIAS ER2017)

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells.The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF)

Adaptación de la distribución minorista al consumidor ecológico: el caso de España y Alemania

El objetivo de este trabajo se centra en el análisis de las diferencias de consumo ecológico en distintos mercados; para ello tratará de determinar las peculiaridades de los perfiles del consumidor verde en España, líder europeo en la producción agrícola ecológica, que puede ser considerado mercado ecológico emergente, y en Alemania, que es el mercado ecológico líder europeo. Además se pretende investigar si los minoristas adaptan sus establecimientos al nivel de importancia de la demanda de productos ecológicos. Los resultados demuestran que los perfiles no cambian demasiado en cuanto a las variables sociodemográficas, y sí lo hacen en variables más personales. Se observa que la oferta de productos ecológicos en España es menor que la de Alemania. Del mismo modo se concluye que la distribución, de forma general, es capaz de adaptar su oferta a la demanda de cada país tanto en términos de área lineal y superficie (m2) como de la variedad de productos

Utilización de crucigramas en la educación a distancia: Un algoritmo para su construcción

En este trabajo se exponen algunos aspectos que caracterizan la educación a distancia, la posibilidad de incluir los crucigramas como una herramienta de motivación para este tipo de enseñanza, se analizan los métodos básicos de construcción de crucigramas y se describe un algoritmo desarrollado para la construcción de estos

Diez meses de análisis de las enfermedades en especímenes silvestres de Litopenaeus setiferus en el Sur del Golfo de México

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90´s. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n~60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNV) and white spot syndrome (WSSV) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presumptive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults.Durante la década de los 90´s se introdujo el camarón blanco del Pacífico Litopenaeus vannamei a los Estados costeros mexicanos del Golfo de México con fines acuícolas, por lo que desde entonces existe preocupación por la posible introducción de enfermedades que puedan afectar a las poblaciones de camarones nativos. La investigación sobre la domesticación de especies nativas para una acuacultura sustentable se ha realizado por más de 10 años, sin embargo, aún existe escasa información sobre las enfermedades que se presentan de manera natural en estas poblaciones y posible trasfaunación. El presente estudio aborda el problema de las enfermedades encontradas en subpoblaciones de jóvenes y adultos de Litopenaeus setiferus del Área natural protegida Laguna de Términos, estado de Campeche, México. Técnicas de histología y biología molecular fueron utilizadas como herramientas de diagnóstico. Se encontró que L. setiferus es portador de patógenos y simbiontes endémicos del Golfo de México, y comparativamente, los jóvenes son más susceptibles en adquirir estas infecciones que los adultos, como probable respuesta al ambiente lacustre que ocupan. No se encontró evidencia de los virus IHHNV y WSSV, aunque en trabajos más recientes en algunos Estados del Norte ya se han detectado en poblaciones silvestres

Nutrient Removal Efficiency of Rhizophora mangle (L.) Seedlings Exposed to Experimental Dumping of Municipal Waters

Mangrove forests are conspicuous components of tropical wetlands that sustain continuous exposure to wastewater discharges commonly of municipal origins. Mangroves can remove nutrients from these waters to fulfill their nutrients demand, although the effects of continuous exposure are unknown. An experimental greenhouse imitating tidal regimes was built to measure the efficiency of mangrove seedlings to incorporate nutrients, growth and above biomass production when exposed to three periodic wastewater discharges. The experiment totaled 112 d. Nutrient removal by the exposed group, such as phosphates, ammonia, nitrites, nitrates and dissolved inorganic nitrogen (97%, 98.35%, 71.05%, 56.57% and 64.36%, respectively) was evident up to the second dumping. By the third dumping, all nutrient concentrations increased in the interstitial water, although significant evidence of removal by the plants was not obtained (p > 0.05). Nutrient concentrations in the control group did not change significantly throughout the experiment (p > 0.05). Treated plants increased two-fold in stem girth when compared to the control (p < 0.05), although control plants averaged higher heights (p < 0.05). Biomass of treated group increased up to 45% against 37% of the control during the duration of the experiment (p < 0.05). We suggest that nutrient removal efficiency of mangroves is linked to the maintenance of oxic conditions in the pore-water because of oxygen transference from their aerial to their subterranean radicular system that facilitates the oxidation of reduced nitrogen compounds and plants uptake. Nevertheless, continuous inflows of wastewater would lead to eutrophication, establishment of anoxic conditions in water and soil, and lessening of nutrient absorption of mangroves

A ten-month diseases survey on wild Litopenaeus setiferus (Decapoda: Penaeidae) from Southern Gulf of Mexico

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90´s. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n~60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNv) and white spot syndrome (WSSv) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presump- tive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults

Pore Water Chemical Variability and Its Effect on Phenological Production in Three Mangrove Species under Drought Conditions in Southeastern Mexico

Mangrove forests have proven to be resilient to most environmental change, surviving catastrophic climate events over time. Our study aimed to evaluate the chemical variability of pore water and its effect on phenological production in three mangrove species (Rhizophora mangle, Avicennia germinans, and Laguncularia racemosa) along the coast of the state of Campeche during a year of severe drought (2009) and a year of average precipitation (2010). Pore water salinity and redox potential were measured monthly in a mangrove forest in 2009 and 2010. Litterfall production and reproductive phenology was measured monthly. We determined the relationships among litterfall production, reproductive phenology, pore water chemistry and precipitation of three species between years. Precipitation, pore water salinity and redox potential significantly differed among years, seasons and sites, and also showed significant interaction between years and seasons (p < 0.05). Significant variation was observed in litterfall production, propagules, flowers, and leaf litter among sites (p < 0.05). A significant change was observed in propagules and flowers among years, and in total litterfall and leaf litter between seasons and species (p < 0.05). Under severe drought, salinity had the strongest effect on total litterfall and propagules in R. mangle, while A. germinans, had the strongest effect on propagule/flower precipitation. Both A. germinans and L. racemosa showed higher resilience than R. mangle at all sites under severe drought conditions. These findings can support activity allocation for mangrove conservation and restoration by providing the tolerance thresholds of the three species that dominate in the regional area of Campeche state. Likewise, this research provides knowledge to the Intergovernmental Experts Group on climate change about drought intensity and its magnitude of impact on mangrove productivity, reproduction and integrity